Cyanobacteria form a phylogenetically coherent group of gram-negative prokaryotes that are capable of oxygenic photosynthesis, wherein their photosystems PSI and PSII extract and transfer electrons from water molecules to electron acceptors and generate oxygen as a co-product. They are capable of fixing carbon from CO2 under aerobic conditions.
As photoautotrophic organisms, the rates of photosynthesis and growth of cyanobacteria are directly affected by the physical parameters of the environment.
In the wild, the competitive success of cyanobacteria depends on a continual fine-tuning of growth rate in order to exploit the changing nutritional environment. To cope with depleted nutrients and exploit those that are plentiful, the cyanobacteria undergo transitions from exponential to arithmetic (linear) growth into non-growth (stationary) physiological states.
The duration of the exponential and linear growth phase in culture depends upon the size of the inoculum, starting density of the inoculum, growth rate, environmental conditions, and capacity of the medium to support microbial growth. Cyanobacterial growth does depend on light intensity. The dependence on external light intensity is impacted by culture density.
It has been reported by Foster that wild cyanobacteria grow optimally in the range of 15-75 μE m−2 s−1 and batch cultures progress from a lag phase into an exponential growth phase. This is typically followed by a period of linear growth that continues until the culture reaches the non-growing stationary phase. Linear growth in bacteria occurs when there are perturbations in the environment such that a critical nutrient is regulated arithmetically. In cyanobacteria, linear growth is most often associated with light limitation caused by self-shading of cells as cultures reach a certain cell density J. S. Foster, et al., Arch. Microbiol, (2007) 187:265-279. The optimal light range may be broader than indicated by Foster, such as 15-300 μE m−2 s−1.
In 1999, Deng and Coleman disclosed the introduction of new genes into the Cyanobacterium Synechococcus PCC 7942 to create a novel pathway for fixed carbon utilization which created the target chemical product ethanol. M.-D. Deng and J. R. Coleman, Appl. Envir. Microbiology (1999) 65: 523-528. Related patents are R. P. Woods, et al. U.S. Pat. No. 6,306,639 and U.S. Pat. No. 6,699,696. Other target chemical products have been identified; see for example, U.S. Pat. No. 7,794,969 and U.S. Pat. No. 8,183,027.
In the production of target chemical products, such as ethanol, from microorganisms, such as cyanobacteria, an inoculum of the microorganism is needed so as to provide a population of such microorganism, suitable for scaling up to levels amenable to commercial scale production. In the case of specialty chemicals, produced in low amounts, this inoculum might be cultured in a vessel so that the cell density increases to a cell density suitable for reaching a production level that meets overall productivity metrics. [See for example PCT/US2011/022790, MICROORGANISM PRODUCTION OF HIGH-VALUE CHEMICAL PRODUCTS, AND RELATED COMPOSITIONS, METHODS AND SYSTEMS; see separately Example 1 of PCT/GB2012/050194] The production of other target molecules from cyanobacteria are discussed in Ruffing et. al., Physiological effects of free fatty acid production in genetically engineered Synechococcus elongatus PCC 7942, Biotechnology and Bioengineering (2012) 109:2190-2199; and in V. H. Work, et al., Biocommodities from photosynthetic microorganisms, Environmental Progress & Sustainable Energy (2013) 32:989-1001. In the case of commodity chemicals, such as biofuels, inoculum scale-up might proceed in several stages.
In the case of inocula to create cultures for open systems, published US application 20100304456 lays out some guidelines:
It is preferred that (1) the amount of biomass provided by the Closed Systems to inoculate the Open Systems should be equal to more than 5% of the carrying capacity of the aggregate Open Systems; (2) the growth rate of the species being cultivated is greater than approximately one and a half doublings per day (i.e. cell biomass doubles about every 16 hours); and that (3) no culture be maintained in any Open System for a period of more than 5 days. The combination of these three limitations assures that, under any circumstances, the culture should attain a biomass of the desired microbe that is equal to at least approximately 90% of the carrying capacity in 5 days or less. This is important for several reasons. First, a culture that is inoculated at a relatively high cell concentration (i.e. greater than 5% of carrying capacity) will dominate the medium compared to any unwanted cells that may have inadvertently been introduced. Second, because most species grow at rates substantially less than 1 doubling every 16 hours (1.5 doublings per day), a species that is capable of growing this rapidly will outpace most potential competitors. Third, the combination of the large inoculum (greater than 5% of carrying capacity) and high growth rate (greater than 1 doubling every 16 hours) assures that, within 5 days, the total biomass will be very near carrying capacity. These conditions are important to (1) reducing the risk of contamination, and (2) promoting the production of total biomass or the biosynthesis or production of oil. First, a potential contaminant would have to have a large inoculum and would have to grow more rapidly than the desired species to dominate the culture medium within 5 days. Second, oil production in particular is favored in cultures that are near carrying capacity because resources become limiting to growth once the culture passes 50% of carrying capacity. By limiting resources favorable to growth, one generally stimulates the biosynthesis of oil.
Paragraph 81 of US 20110217692 shows the risks of contamination.
Paragraph 82 of US 20110287541 discusses amounts stored for use as inocula.
Example 4 of PCT/AU2011/000829 describes inoculation of a large bioreactor with a volume of inoculum, followed by growth, and followed by further dilution.
Earlier art mentions the preparation of inocula ultimately for use in open, rather than closed, systems. For example, H. W. Blanche, Current Opinion in Biotechnology (2012) 23:390-395; E. Olguin, Biotechnology Advances (2012) 30:1031-1046; J. Quinn, Bioresource Technology (2012) 117: 164-171; I. Christenson, Biotechnology Advances (2011) 29:686-702 [discussing Cellana].
The present invention is directed to the creation of inocula suitable for introduction into closed bioreactor systems. The present invention provides a method for rapid scale-up of inoculum by monitoring of optical density, and control thereof. An embodiment of the invention permits the method to proceed by minimizing exposure of the inoculum to ambient air.
In another embodiment, a plurality of photobioreactors connected in parallel can be inoculated from a series of scale-up cultures. In a further embodiment, the culture is transferred rapidly and evenly to the plurality of photobioreactors. In a further embodiment, the process is performed so that the photobioreactor culture is axenic or substantially axenic.